We have recently shown that adult MZ B cells contain 20% of sequences that lack N region diversity, a hallmark of fetally-derived cells. Through the use of mixed bone marrow chimeras comprised of TdT+ and TdT- bone marrow cells, we have demonstrated that the differences in repertoire that we observed between MZ and FO B cells was greatly influenced by BCR-based selection into these subsets. During the analyses of these mice, we observed unexpected and striking differences in the repertoires of all subpopulations of B cells from immature B cells in the bone marrow through the mature B cells in the spleen. Due to the similarity in the selective preferences for N- IgH sequences and also for lambda light chains between the transitional T1 compartment and MZ B cells in the bone marrow chimeras, we propose that a subset of MZ B cells with a distinct repertoire may have directly differentiated from the T1 transitional compartment without going through the T2 stage. We thus hypothesize that the MZ compartment may be comprised of distinct subsets with distinct repertoires, and we will focus on this novel hypothesis in this grant. We have recently identified a mouse that has only the subset of MZ cells that directly differentiates from T1 cells, providing us with a unique approach to the studies proposed in this application. The experiments presented here will characterize the multiple MZ precursor populations and novel pathways of B cell differentiation in the spleen that we believe we have uncovered. We will assess the differences in the repertoires of these subsets of MZ and FO B cells with regard to microbial, protein, autoantigen and glycan specificities using microarrays. We will also assess how these subsets and these repertoires change with age, since one of the MZ subsets appears to have a fetal origin. This information of potentially different and possibly changing repertoires in different splenic subsets will be critical information for vaccine design strategies that may target MZ B populations, and for understanding the response to blood-borne pathogens at different stages of life. PUBLIC HEALTH RELEVANCE: Marginal zone B cells make rapid responses to blood-borne pathogens, including many bacteria. We propose that the marginal zone B cells can be subdivided based on BCR repertoire differences, cell surface phenotype, and differentiation history. We will analyze the antibody repertoire of these subsets of marginal zone B cells to autoantigens, glycans, and bacterial pathogens, and will determine if the repertoire changes with age. This information of potentially different and possibly changing repertoires in different splenic subsets will be critical information for vaccine design strategies that may target MZ B populations, and for understanding the response to blood-borne pathogens at different stages of life.